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P.C. Montgomery, N.L. O'Sullivan; Development of Immortalized Rat Conjunctival Epithelial Cell Lines: An in vitro Model to Examine Transepithelial Antigen Uptake . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4928.
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© ARVO (1962-2015); The Authors (2016-present)
To develop conjunctival epithelial cell lines for investigation of antigen translocation across a mucosal barrier.
Rat conjunctival epithelial cells were grown in serum–free medium and immortalized using the SV40 large T antigen. Longevity and doubling times were monitored in continuous culture. Cytokeratin expression patterns and tight junction formation were determined using immunocytochemistry. Barrier function was assessed by transepithelial electrical resistance (TER) measurements and translocation of soluble and particulate antigens across the epithelial layer using a Transwell culture system.
Conjunctival epithelial cells from Fischer 344 rats were immortalized with pSV3(neo) resulting in two cell lines – CJ4.1A and CJ4.3C. Each formed confluent cell layers with epithelial morphology when grown on permeable membrane filters. They expressed the SV40 T antigen, the conjunctiva–specific cytokeratin 4, the goblet cell–specific cytokeratin 7 and were negative for the corneal epithelial cell–specific cytokeratin 12. Population doubling times were 20.2 ± 1 h for CJ4.1A and 23 ± 4 hrs for CJ4.3C. The cell lines have been in culture for over 60 (CJ4.1A) or 40 (CJ4.3C) passages. When grown on TranswellTM membranes, each cell line achieve a transepithelial electrical resistance of more than 1000 Ω cm2 by 3–4 days and maintained this high resistance for several days. At 24h following addition of 500 µg/ml of FITC–labeled ovalbumin to the apical chambers, 0.6 ± 0.1 µg/ml could be detected in the basal chamber of CJ4.1A and none in the basal medium of CJ4.3C. In contrast, 56 ± 0.6 µg/ml was detected in the lower chambers of cell–free Transwells. Similarly, Transwells containing confluent CJ4.1A or CJ4.3C cells impeded passage of 0.1 µl polystyrene microspheres (7.0 ± 0.5 and 5.6 ± 0.5 percent of the apical input, respectively), compared to 44.2% of the input microspheres recovered from the basal chambers of wells with no cells.
Cell lines have been established and a cell culture model has been developed to investigate the translocation of protein and particulate antigen across the conjunctival epithelial barrier. Morphological and functional characterization show that these cells will be a useful experimental tool to assess strategies to enhance transepithelial antigen uptake.
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